CN117604027B - Novel efficient genetic transformation method for pineapple - Google Patents
Novel efficient genetic transformation method for pineapple Download PDFInfo
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Abstract
The invention provides a novel efficient genetic transformation method for pineapple, which comprises the steps of inducing pineapple callus, carrying out dark culture proliferation, continuing dark culture, inducing adventitious bud differentiation of pineapple callus through dark culture, enabling rosette-shaped cluster buds to extract stems, carrying out agrobacterium infection according to a section cut (1 section/section) serving as a receptor material, carrying out redifferentiation and screening to obtain transgenic plants, and thus, the efficiency of agrobacterium tumefaciens mediated transformation is remarkably improved, the positive rate of PCR detection of genetic transformation plants can reach 41.51%, which is remarkably higher than that of the existing method, and the method has wide application prospect in pineapple large-scale transgenic practice.
Description
Technical Field
The invention relates to a genetic transformation method, in particular to a novel efficient pineapple genetic transformation method.
Background
Pineapple (Ananas comosus), also known as pineapple, is one of the three tropical fruit trees in the world, and is also an important ornamental plant and fiber plant. Like other horticultural crops, germplasm resource creation and variety improvement of pineapple still rely on conventional breeding techniques such as cross breeding, mutation breeding and bud mutation seed selection. As pineapple is a asexual propagation plant, the pineapple has narrow genetic foundation, high genome heterozygosity, strong gene linkage and low genetic variation rate, and a germplasm material with excellent genetic characters is difficult to create by a conventional breeding technology. On the other hand, pineapple is a strict gametophyte type self-incompatibility plant, homozygotes cannot be obtained through self-hybridization, and great trouble is caused to gene positioning and genetic rule analysis.
Along with rapid development of bioscience, methods of gene analysis function identification, gene editing, transgenic breeding, intelligent design breeding transformation and the like are gradually introduced into plant breeding research, new varieties of transgenic corn, soybean, cotton and rape with herbicide resistance, insect resistance and the like are cultivated by utilizing a transgenic technology, and large-area global planting is performed since 1996, so that great economic and social benefits are generated. However, these studies must be based on efficient genetic transformation systems. Genetic transformation of pineapple began at the end of the last century, firoozabady et al (1998, 1999) introduced GUS gene into pineapple plants by Agrobacterium tumefaciens mediated method using embryogenic callus as the recipient material for transformation. Thereafter, genetic transformation of pineapple has been reported (Firoozabady et al 2005; firoozabady & Guterson 1998; firoozabady & Moy 2004; he Yehua et al 2006; jun et al 2012;Ming,2018;Sripaoraya et al, 2001).
Although the existing pineapple in-vitro regeneration technology is mature (He Yehua and the like, 2006,2010,2012), due to the uniqueness of pineapple and the like, the establishment of a stable pineapple transformation system is still difficult for researchers with less experience accumulation of transgenic technology, and the acquisition of transgenic materials capable of changing genetic traits is quite few. In prior reports, pineapple transformation recipients have been used for stem tips, leaf bases, callus, suspension cell lines, somatic embryos, and the like (He Yehua et al, 2006; wang et al, 2009; xie Tao, 2019), however, pineapple transformation efficiencies are still low at present. The cumulative positive rates after 3, 5 and 7 passages of screening were 9.9%, 0.77% and 0.017% respectively using normal calli as transformation recipients (Fang, 2009). Therefore, the search for new receptor materials and transformation methods to increase pineapple transformation efficiency is of great significance in promoting the application of transgenic technology thereof.
The efficient genetic transformation system of plants must first rely on good acceptor materials, which should be tissues that are available in large quantities, have a strong plant regeneration capacity and are easily accessible for T-DNA introduction. Because the receptor material needs to produce wounds and is easy to regenerate when being mediated by agrobacterium, in the currently used pineapple transformation material, the stem tip is not suitable to be directly used as the receptor material of the transgene because of the small quantity, laborious sterilization and difficult redifferentiation; on the other hand, since pineapple is easy to differentiate, callus, suspension cell line, somatic embryo and the like cultured for more than 4-5 generations are observed under a stereoscopic vision, and are mainly composed of rosette-shaped (without obvious stems) buds to form clustered adventitious buds, and the transformation efficiency of taking clustered buds as receptor materials is very low; while leaf bases are slightly more efficient as transformation receptors, they must be young leaf bases derived from tissue culture to produce adventitious buds, and are limited in number.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a novel efficient pineapple genetic transformation method.
The technical scheme of the invention is as follows:
a pineapple high-efficiency genetic transformation method comprises the following steps:
(1) Callus induction: inducing pineapple non-embryogenic callus, and then culturing and proliferating in dark;
(2) Stem tissue culture: dark culture is continued until stalks are generated on the pineapple calluses;
(3) Plant transformation: the nodes of pineapple stems are used as incisions to cross the pineapple stems, the pineapple stems are divided into a plurality of sections to generate wounds, and then activated transgenic agrobacterium is adopted for infection;
(4) Screening and culturing: inoculating the infected pineapple stalk stem sections into culture mediums containing Km with different concentrations for multiple rounds of screening, and finally transferring the resistant buds to rooting culture mediums for rooting culture;
(5) Hardening and transplanting to obtain normal pineapple seedlings;
(6) And (5) detecting PCR positive detection.
Wherein, the culture medium for dark culture in the step (1) is MS+2.8-3.2 mg/L6-BA+1.8-2.2 mg/L NAA, preferably MS+3.0 mg/L6-BA+2.0 mg/L NAA.
Wherein, the culture medium for dark culture in the step (2) is MS+2.8-3.2 mg/L6-BA+1.8-2.2 mg/L NAA, preferably MS+3.0 mg/L6-BA+2.0 mg/L NAA.
Wherein the temperature of the dark culture in step (1) is 25 to 32 ℃, preferably 26 to 30 ℃, more preferably 28 ℃.
Wherein the temperature of the dark culture in the step (2) is 25 to 32 ℃, preferably 26 to 30 ℃, more preferably 28 ℃.
Preferably, in step (2) the cultivation is performed in the dark until up to more than 10cm, with more than 8 knots of stalks are produced on the pineapple calli.
Preferably, the dark culture in step (2) is carried out for 50 to 70 days, preferably 58 to 72 days, more preferably 60 days.
Preferably, in step (4) 4 rounds of anti-Km screening, km concentrations are 28-32mg/L, 38-42mg/L, 48-52mg/L and 58-62mg/L in order, preferably Km concentrations are 30mg/L, 40mg/L, 50mg/L and 60mg/L in order.
More preferably, in the step (4), the stem segments of pineapple stalks after infection are inoculated to the screening medium S1 for normal culture, and then the culture medium is changed after 26-30d culture in each medium according to the inoculation to the screening medium S2, the screening medium S3 and the screening medium S4.
Wherein, the screening culture medium S1 is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose+48-52 mg/LCarb +28-32mg/L Kan, preferably MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+50 mg/L Carb+30mg/L Kan,
Wherein the screening culture medium S2 is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose+38-42 mg/L Carb+38-42mg/L Kan, preferably (MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+40 mg/LCarb +40mg/L Kan,
Wherein, the screening culture medium S3 is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose+28-32 mg/LCarb +48-52mg/L Kan, preferably MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+30 mg/L Carb+50mg/L Kan,
Wherein, the screening culture medium S4 is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose+18-22 mg/L Carb+58-62mg/L Kan, preferably MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+20 mg/LCarb +60mg/L Kan,
Wherein, the rooting culture medium is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose, preferably MS+1.0mg/L IBA+1.0mg/LNAA+7g/L agar+30 g/L sucrose.
Preferably, in step (3) the transected stem segments are pre-cultivated in the dark for 2-3d before transfection, the pre-cultivated medium being: MS+6-BA 1.8-2.2mg/L+NAA0.9-1.1 mg/L+sucrose 25-35 g/L+agar 5-9g/L, preferably MS+6-BA2mg/L+NAA 1 mg/L+sucrose 30 g/L+agar 7g/L.
Wherein, after the infection in the step (3) is finished, the pineapple stem segments are co-cultivated with agrobacterium-based darkness for 2-3d.
Wherein, the culture medium for co-culture is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8g/L agar+28-32 g/L sucrose+90-110 mu mol/L AS, preferably MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+100 mu mol/L AS.
The transgenic agrobacterium is agrobacterium containing target gene, which can be selected by the person skilled in the art according to practical application, and the invention is not limited in particular. In one embodiment of the present invention, the transgenic agrobacterium is selected from agrobacterium tumefaciens GV3101 containing pK7WG2D-AcPI, and it should be understood that the present invention is not limited thereto, and one skilled in the art may select other target genes to be linked to a suitable vector according to actual needs to transform into transgenic agrobacterium for application.
The invention establishes a new pineapple high-efficiency genetic transformation method, the adventitious bud differentiation of pineapple callus is induced through dark culture, rosette-shaped cluster buds are used for extracting light stems, then agrobacterium infection is carried out according to section cutting (1 section/section) as a receptor material, transgenic plants are obtained through re-differentiation and screening, the agrobacterium tumefaciens mediated transformation efficiency is remarkably improved, the positive rate of PCR detection genetic transformation plants can reach 41.51 percent, which is remarkably higher than that of the existing method, and the method has wide application prospect in pineapple large-scale transgenic practice.
Drawings
FIG. 1 shows that pineapple callus develops into a stem by dark culture.
FIG. 2 shows the induction of callus and adventitious buds in pineapple stem segments under light and darkness.
FIG. 3 shows the rooting and transplanting conditions of pineapple stalk and stem segments inoculated in different screening media after infection. A: s1, culturing. B: s2, culturing. C: s3, culturing. D: s4, culturing. E: rooting culture. F: the transgenic plants grow after transplanting.
FIG. 4 shows the results of transgene identification. A: acActin primer detection. B: kan primer detection. C: vector forward and gene backward detection. D: and C, comparing the PCR product detected as the genetic transformation positive plant with the sequence of the transgenic vector.
FIG. 5 is a phenotype observation of transgenic plants. A. B, C: wild type 'shenwan'. D. E, F: transgenic 'shenwan'.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1 Material
The pineapple material for the test is 'shenwan' (Ananas comosus cv. Shenwan), which is obtained from an orchard of the university of agricultural university of south China, and the suction buds of the pineapple material are taken as explants; plant overexpression recombinant plasmid (pK 7WG 2D-AcPI) containing AcPI (see Chinese patent application CN202210519193.6, preparation method, "a functional gene AcPI for regulating plant organ morphology and application thereof").
It should be understood that the experiment is performed by using "shenwan" as pineapple material, and other pineapple varieties may be used as materials for performing related experiments, such as 'ba li', 'yu exquisite', 'rock candy red', 'red pearl', 'hot purple leaf', 'madder bi', and so on.
2 Methods and results
2.1 Callus induction
The induced pineapple was grown in dark culture at 28℃after callus formation according to the method of He Yehua et al (He Yehua et al, 2010). The specific operation is as follows:
in an ultra-clean workbench, the leaf blades of aseptic seedlings (3-5 cm high) are torn off with basal stems, non-embryogenic callus is induced on MS+2.0mg.L -1 6-BA+2.5mg·L-1 NAA culture medium, and after 4 weeks, the non-embryogenic callus is cut off and transferred to proliferation culture medium (MS+3mg.L -16-BA+2mg·L-1 NAA), and callus proliferation culture is carried out every 4 weeks for 1 time.
2.2 Stem tissue culture
After inducing the callus, the pineapple callus is continuously dark-cultured in dark (28 ℃) for about 60 days (without changing culture medium), a plurality of thin and long stems are emitted on the pineapple callus at the moment, and agrobacterium-mediated pineapple genetic transformation is carried out by using the stems.
Pineapple (A. Comosus) callus was cultivated in the dark (28 ℃) for about 60 days without changing the medium, and the callus was allowed to slowly grow into stalks (FIG. 1). Pineapple stalks are cultivated to a position of 2/3 of a tissue culture bottle, when the thickness of the stalks is about 1.5mm, the stalk tissues are cut into 1cm stalk segment tissues on an ultra-clean workbench (note that the stalk nodes are necessarily cut, growing points are arranged at the positions), the stalk tissues are orderly arranged on an MS solid culture dish, the pineapple stalks are respectively cultivated under light and dark for about 30d, the pineapple stalks are slowly differentiated under the light to form adventitious buds, interestingly, the callus is induced under the dark cultivation, and plant tissues similar to pineapple leaves are naturally also grown (figure 2), so that the stalk segments have the capability of differentiating the callus and are suitable for being used as receptors for transforming pineapple.
The advantages are readily apparent when the recipient is a culture of callus, suspension cell lines, somatic embryos or the like in vitro, which is readily available in large quantities, sterile, and does not require dedifferentiation. However, when the callus formed by dedifferentiation of pineapple is transferred on a proliferation medium (MS+3.0 mg/L BA+2.0mg/L NAA) 3 times or more, a large number of small bulb-like adventitious buds with a diameter of about 1mm are produced. The sections were observed under the stereoscope and these bulbs were actually rosette-like, with 1-several young leaves as the coating, with a hollow middle and very fine top growth points. Such small bulbs are less likely to have T-DNA enter the recipient cells than they would have been if they were stained without cutting from the growing point. The suspension cell line just taken from the liquid culture medium is too dispersed to cut out wounds easily, and needs to be cultured on a solid culture medium for 1 generation. The somatic embryo needs to be embryogenic callus at the initial stage of somatic embryo induction, and the material conversion rate of the somatic embryo entering the middle and later stages of somatic embryo differentiation is low. However, in the invention, the pineapple stem is used for culturing the callus for transformation experiments, so that the availability and freshness of the receptor are greatly improved, and the transformation efficiency is greatly improved. The stem tissue which is obtained by adopting the 1 generation callus obtained by the explant and is developed again is taken as a receptor, which is one of secret tables of successful transformation.
2.3 Plant transformation
Transformation was performed according to the method of He Yehua et al (He Yehua et al 2012), and Agrobacterium tumefaciens GV3101 containing pK7WG2D-AcPI was streaked, and single-clone was picked up in YEP liquid medium containing Kan (100 mg/ml), and shaken overnight in a 28℃shaker (280 rpm) to a bacterial solution concentration of 0.5-0.8 ng/. Mu.L. When stem tissue grows to 2/3 of a tissue culture bottle (the height is more than 10cm and the stem has more than 8 knots), pineapple stems are carefully transected by a dissecting knife in an ultra-clean workbench, the incision is a node (the incision is provided with a growing point for differentiation and healing), and the tissue culture bottle is divided into a plurality of sections, and wounds are generated for agrobacterium infection. The stem tissue was cut into 1cm size and inoculated into a medium of MS+6-BA2mg/L+NAA1 mg/L+sucrose 30 g/L+agar 7g/L, pH=5.8, and pre-cultured in the dark for 2d. Transferring the pre-cultured pineapple stem segments into a 20mL sterile injector, adding 10m L agrobacterium tumefaciens bacteria liquid into the sterile injector, pushing an injector core rod to discharge redundant gas to the 10mL scale of the injector, plugging an injection head by a rubber plug, and pulling the core rod to the 20mL scale to enable the agrobacterium tumefaciens to be in a vacuum environment to infect the pineapple stem segments, wherein each vacuum infection is carried out for 1.5min, and the injector is continuously rocked during the period. After infection, co-culture is carried out for 2-3d under dark conditions under MS+2.0mg/L6-BA+1mg/L NAA+7g/L agar+30 g/L sucrose+100. Mu. Mol/L AS. After co-cultivation, taking out pineapple stem segments, washing with sterile water for 3 times, washing off agrobacterium on the surface, inoculating the stem segments to a screening culture medium S1 for cultivation, starting differentiation of callus and adventitious buds after 10d, transferring the callus and the adventitious buds to screening culture media S2, S3 and S4 after 28d, sequentially culturing in each culture medium for 26-30d, and then transcribing green buds to a rooting culture medium (finishing screening culture and rooting culture under illumination).
Screening culture medium S1 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30g/L sucrose+50mg/L Carb+30mg/L Kan,
Screening culture medium S2 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30 g/L sucrose+40 mg/L Carb+40mg/L Kan,
Screening culture medium S3 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30 g/L sucrose+30 mg/L Carb+50mg/L Kan,
Screening culture medium S4 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30 g/L sucrose+20 mg/L Carb+60mg/L Kan,
The rooting culture medium is MS+1.0mg/L IBA+1.0mg/LNAA+7g/L agar+30g/L sucrose.
The results showed that agrobacterium-mediated pK7WG2D-AcPI transformed 1540 stem segments, and 53 anti-Kan plants were obtained after 4 consecutive rounds of selection. After the second round of screening culture, the two ends of pineapple stem segments begin to differentiate callus, part of stem segment tissues are blackened, part of stem segment axillary buds begin to differentiate adventitious buds, callus is left, blackened tissues and axillary bud tissues are eliminated, and the occurrence rate of pAcPI second-generation callus is 83.1% (table 1). And S3, after the second round of selective culture, the callus in the previous stage grows adventitious buds, 2 to 5 leaves which can be distinguished by naked eyes can be seen, and the green bud (Kan resistant bud) rate is 56.2 percent. Continuously increasing Kan mass concentration to 60mg/L, and increasing selection pressure, so that most false positive plants in round 1 are removed due to reduction to white plants after the 4 th round of selection culture is finished; the elimination rate is 63-78%. After the 4 th round of selection, transferring the resistant buds into a rooting culture medium, when the plants are cultured to have more than 8 leaves with the length of about 3cm and more than 5 roots with the length of more than 1cm, hardening off and transplanting can be carried out, and the transformed seedlings can grow normally after transplanting.
TABLE 1pAcPI screening cases for different Kan concentrations
2.4 Molecular detection of resistant plants
Respectively taking 0.2g of transformed plant and non-transformed plant leaves, extracting DNA by adopting a CTAB method, extracting 53 green pineapple tissue culture seedling genome DNA, detecting the DNA by using an action according to AcActin upstream primers (AcActin-F: CTGGCCTACGTGGCACTTGACTT, ACACTIN-R: CACTTCTGGGCAGCGGAACCTTT), carrying out PCR amplification on the genome DNA by using a Kan primer (Kan-F: GTTCTTTTTGTCAAGACCGACC, KAN-R: CAAGCTCTTCAGCAATATCACG), and finally carrying out PCR amplification on the target fragment of the genome DNA by using an AcPI specific primer and a vector universal primer (35S-F: CTATCCTTCGCAAGACCCTTC, QPI-R: CCCTCATGTTCCCATTCATC). The above was detected by using I-5 2 Xhigh-FIDELITY MASTER Mix of the Prime bioscience technology, and the PCR amplified product was detected by 1% agarose gel electrophoresis.
As a result, as shown in FIG. 4, 50 of 53 green pineapple seedlings were amplified to obtain a target fragment (FIG. 4-B) by PCR amplification of the genomic DNA using the action primer (FIG. 4-A), and 22 of 53 obtained target fragments were amplified by PCR amplification of the genomic DNA using the AcPI specific primer and the vector universal primer, respectively, 13, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 47-1, 48, 49 plants in FIG. 4-C, and the genetic transformation positive rate was 41.51% by PCR product detection and sequence comparison identification (FIG. 4-D).
2.5 Phenotypic outcome
Transgenic plants (FIGS. 5D-F) showed a special appearance of red color, except for the number of covered hair scales on the leaf base and leaf surface, compared to wild type plants (FIGS. 5A-C).
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (6)
1. A pineapple genetic transformation method, which is characterized by comprising the following steps:
(1) Callus induction: inducing pineapple non-embryogenic callus, and then carrying out dark culture proliferation at 28 ℃, wherein the culture medium for dark culture is MS+2.8-3.2 mg/L6-BA+1.8-2.2 mg/L NAA;
(2) Stem tissue culture: dark culture is continued for 50-70 days at 28 ℃ until more than 10cm and more than 8 sections of stalks are generated on pineapple callus, wherein the culture medium is MS+2.8-3.2 mg/L6-BA+1.8-2.2 mg/L NAA;
(3) Plant transformation: cutting a stem tissue of pineapple into 1 cm pieces by taking a node of the stem of pineapple as a notch, inoculating to a culture medium of MS+6-BA 2 mg/L+NAA1 mg/L+sucrose 30 g/L+agar 7 g/L, pre-culturing in the dark for 2d pieces, and then infecting with activated transgenic agrobacterium; after infection, co-culturing pineapple stem segments and agrobacterium-based darkness for 2-3d; the co-culture medium is MS+1.8-2.2 mg/L6-BA+0.8-1.2 mg/L NAA+6-8 g/L agar+28-32 g/L sucrose+90-110 mu mol/L AS;
(4) Screening and culturing: after co-cultivation is finished, taking out pineapple stem segments, washing the pineapple stem segments with sterile water for 3 times, washing out agrobacterium on the surface, inoculating the pineapple stem segments to a screening culture medium S1 for cultivation, starting differentiation of calluses and adventitious buds after 10 d, transferring the calluses and the adventitious buds to screening culture media S2, S3 and S4 after 28 d, sequentially culturing the calluses and the adventitious buds in each culture medium for 26-30 days, transcribing green buds into a rooting culture medium, and finishing screening culture and rooting culture under illumination conditions;
screening medium S1 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30g/L sucrose+ mg/L Carb+ mg/L Kan,
Screening medium S2 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30g/L sucrose+ mg/L Carb+ mg/L Kan,
Screening medium S3 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30g/L sucrose+ mg/L Carb+ mg/L Kan,
Screening medium S4 is MS+2.0mg/L6-BA+1mg/LNAA+7g/L agar+30g/L sucrose+ mg/L Carb+60 mg/L Kan,
The rooting culture medium is MS+1.0mg/L IBA+1.0mg/LNAA+7g/L agar+30g/L sucrose;
(5) Hardening and transplanting to obtain normal pineapple seedlings;
(6) And (5) detecting PCR positive detection.
2. The pineapple genetic transformation method of claim 1, wherein in step (1), the medium for dark culture is ms+3.0 mg/L6-ba+2.0 mg/L NAA;
The medium for the dark culture in the step (2) is MS+3.0 mg/L6-BA+2.0 mg/L NAA.
3. The pineapple genetic transformation method of claim 1 or 2, wherein the pineapple is dark cultured for 58-72 days in step (2).
4. A pineapple genetic transformation method according to claim 3, wherein the dark culture in step (2) is for 60 days.
5. The method for genetic transformation of pineapple according to claim 1, wherein in the step (3), the co-culture medium is MS+2.0 mg/L6-BA+1 mg/L NAA+7g/L agar+30 g/L sucrose+100. Mu. Mol/L AS.
6. The pineapple genetic transformation method of claim 1, wherein the transgenic agrobacterium is agrobacterium tumefaciens GV3101 containing pK7WG 2D-AcPI.
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